Glycated protein is a non-enzymatically-glycated protein, which is produced as a result of covalent bonding between an aldehyde group of sugar, namely, aldose (i.e., a monosaccharide potentially associated with an aldehyde group, and derivatives thereof), and an amino group of a protein. Examples of the amino group of the protein include an N-terminal α-amino group and an internal lysine residue side chain ε-amino group. These glycated proteins are also referred to as Amadori compounds for being formed upon Amadori rearrangement of a Schiff base, a reaction intermediate product.
The glycated proteins are contained in body fluid such as blood in body or in a biological sample such as hair. The concentration of the glycated protein in blood strongly depends on the concentration of saccharide such as glucose dissolved in serum. In diabetic conditions, production of the glycated protein is accelerated. A concentration of glycated hemoglobin in erythrocytes and a concentration of glycated albumin in serum indicate an average blood sugar level for the past predetermined period. Therefore, quantification of the glycated proteins is important for diagnosis and control of the disease process of diabetes.
Examples of known conventional methods for quantifying a glycated protein include, for example, a method employing high-performance liquid chromatography (Chromatogr. Sci., 10, 659 (1979)), a method using a column loaded with a solid material bound with boric acid (Clin. Chem., 28, 2088–2094 (1982)), a method employing electrophoresis (Clin. Chem., 26, 1598–1602 (1980)), a method using antigen-antibody reaction (JJCLA, 18, 620 (1993)), a colorimetric method for determining reducibility using tetrazolium salt (Clin. Chim. Acta, 127, 87–95 (1982)), a calorimetric method using thiobarbituric acid following oxidation (Clin. Chim. Acta, 112, 197–204 (1981)), a method using an enzyme such as glycated amino acid oxidase (Japanese Patent Examined Publication (kokoku) No. 05-33997, Japanese Patent Application Laid-Open (kohyo) No. 11-127895, WO97-13872, Japanese Patent Examined Publication (kokoku) No. 6-65300, and Japanese Patent Applications Laid-Open (kohyo) Nos. 2-195900, 3-155780, 4-4874, 5-192193, 6-46846, 11-155596, 10-313893, 11-504808, 2000-333696, 2001-54398, 2001-204495 and 2001-204494). Furthermore, a new method for quantifying a glycated protein was disclosed recently which is more accurate than any of the above-mentioned methods (Japanese Patent Application Laid-Open (kohyo) No. 2001-95598). According to this quantification method, a sample containing a glycated protein is treated with protease to release a fructosyl peptide from the glycated protein, which is then exposed to oxidase. The resulting product is quantified for quantification of the glycated protein. This method has been recognized as an accurate quantification method that requires short time and simple manipulation.
According to the conventional method using glycated amino acid oxidase, a glycated protein is treated with protease and then the produced glycated amino acid is quantified enzymatically. Specifically, according to this method, a glycated protein is treated with protease or the like to give fructosyl amino acid, which is then exposed to fructosyl amino acid oxidase to quantify the produced hydrogen peroxide. Examples of known fructosyl amino acid oxidases that can be used in such quantification method include oxidase produced by Corynebacterium (Japanese Patent Examined Publications (kokoku) Nos. 5-33997 and 6-65300), oxidase produced by Aspergillus (Japanese Patent Application Laid-Open (kohyo) No. 3-155780), oxidase produced by Gibberella (Japanese Patent Application Laid-Open (kohyo) No. 7-289253), oxidase produced by Fusarium (Japanese Patent Applications Laid-Open (kohyo) Nos. 7-289253 and 8-154672), oxidase produced by Penicillium (Japanese Patent Application Laid-Open (kohyo) No. 8-336386), oxidase produced by Tricosporon (Japanese Patent Application Laid-Open (kohyo) No. 2000-245454), and ketoamine oxidase (Japanese Patent Application Laid-Open (kohyo) No. 5-192193). However, while these enzymes act well on fructosyl amino acids, they do not act on fructosyl peptides. Oxidase produced by E. coli DH5α (pFP1) (FERM BP-7297) described in Japanese Patent Application Laid-Open (kohyo) No. 2001-95598 is known as an enzyme that acts on fructosyl peptide. However, preparing a quantification kit with this enzyme is difficult since its basic physicochemical properties are unknown and it has poor stability.
The objective of the present invention is to provide a novel and stable fructosyl peptide oxidase, a fructosyl peptide oxidase gene encoding the same, and a method for producing the novel fructosyl peptide oxidase.